The conformation of chromatin is a primary mechanism by which the cell regulates DNA-templated processes. Consistent with this broad role, aberrations in the enzymes and structural components responsible for controlling chromatin dynamics have been linked to an extensive range of human diseases, including the majority of cancers and an increasing number of genetic and developmental disorders. Recent technological advancements have increased our knowledge of how the positions of nucleosomes on DNA are regulated and how chromatin forms large three-dimensional (3D) loops called chromatin domains. 3D chromatin structure is hypothesized to be capable of both promoting and inhibiting transcription depending on context. However, understanding the mechanisms and functions of these types of chromatin structures has been difficult due to the low resolution of current methods, which have made it almost impossible to determine chromatin structure within cells at scales necessary to determine its relationship to the expression of single-genes. As a result, the long-held hypothesis that 3D chromatin structure at this level regulates transcription has been largely untested in a physiological context. To examine 3D chromatin structure in cells in which it is expected to function extensively, the candidate has implemented a genomics method capable of mapping 3D chromatin structure genome-wide at unprecedented single-nucleosome 150 base pair resolution in quiescent S. cerevisiae. Quiescent yeast bear conserved hallmarks of quiescent cells, in particular widespread transcriptional repression and chromatin condensation, which make them an excellent model for determining the mechanisms by which 3D chromatin structure represses transcription. Preliminary results have led to the hypothesis that in quiescent cells, the condensin complex represses transcription by inducing quiescence-specific 3D chromatin structures.
Aim 1 of this proposal will determine how condensin is targeted to form chromatin domains during quiescence using genomics, microscopy, and a single-molecule magnetic tweezer assay.
Aim 2 will examine the conformation of chromatin within domains to determine if it is folded into 3D structure at a smaller scale and investigate whether chromatin structure at this scale is the mechanism by which transcription is repressed during quiescence. The mentored component of this work will be completed under the sponsorship of Dr. Toshio Tsukiyama, an expert in the chromatin field, at one of the premier institutes for basic science and cancer research, the Fred Hutchinson Cancer Research Center. The candidate will also be trained in single- molecule biochemical assays under the supervision of Dr. Sue Biggins, and will expand her proficiency in bioinformatics through coursework and independent study. This research and training will provide the candidate with an exciting model system and the skills necessary for a successful independent career.
This project will increase our knowledge of the mechanisms and functions of chromatin, which has been implicated in the majority of cancers and in many genetic and developmental disorders. It will also investigate the mechanisms of cellular quiescence, which is a process that has been linked to conferring drug resistance to cancer cells. These studies will aid researchers in the field and have the potential to identify new targets for anti-cancer therapies.
